A rapid and sensitive method to determine the relative specificity (τ) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) using anion-exchange chromatography is described. We employed the open gas system for the reaction of RuBisCO to get the most reliable CO2 and O2 concentrations in the reaction mixture. 3-Phosphoglycerate and 2-phosphoglycolate which were formed in the RuBisCO reaction were completely separated and directly measured with anion-exchange chromatography without using radioisotopes. The determinaton of the τ value was accomplished in 3 h. The τ values of RuBisCO enzymes from higher land plants were between 90 and 96, and those from bacteria including cyanobacterium were close to 45. These values were in agreement with previously reported values. The enzyme of the red macroalga Porphyra yezoensis exhibited a τ value of over 140, as expected from the reported value of the enzyme from the red microalga Porphyridium cruenteum. RuBisCO from the green macroalga Ulva pertusa had a τ value close to 70 and similar to that of the enzyme from the green microalga Chlamydomonas reinhardtii.